One of the major challenges of HIV-1 vaccine development is the elicitation of broadly neutralizing antibodies (bNAbs) against HIV Env. The recent isolation of several bNAbs from HIV-infected individuals demonstrates that the human B cell repertoire can generate bNAbs targeting the conserved Env region. However, there is still a tremendous knowledge gap regarding how the broad responses are elicited during chronic HIV-1 infection and, additionally, how these compare to the much more limited responses elicited by Env vaccination. To fill this information gap, we propose to define and improve the elicitation of neutralizing antibodies toward the most functionally conserved and accessible element of the HIV-1 Env complex, namely the CD4 binding site (CD4bs), which is crucial for virus engagement with receptor CD4. Previously, we developed a multicolor Env epitope-specific B cell sorting and RT-PCR strategy to exploit the memory B cell compartment of HIV-infected individuals, which led to the isolation of CD4bs bNAb, VRC01, which neutralizes >90% of circulating primary viruses. Interestingly we find that VRC01 and CD4i antibody subsets can mutually enhance binding affinity for gp120, suggesting that CD4i MAbs may be associated with the evolution of VRC01-like bNAbs during the natural infection process. We adapted this epitope-specific memory B cell sorting strategy to extend our study to gain insight of Env B cell response, which is summarized as follows. (1) To characterize the neutralizing antibody response elicited by vaccination of Env-inoculated non-human primates (NHPs). We will perform memory B cell FACS sorting, clonal analysis and IgG gene deep sequencing of Env-specific B cells. (2) To determine the longitudinally evolving neutralizing antibody response in selected HIV-infected individuals. We will investigate factors associated with bNAb response from a few highly selected HIV-1-infected individuals by analyzing their CD4bs bNAbs and their complementary antibodies including CD4i antibodies. (3) To advance immunogen design by examining if i) Env-CD4i MAb complex as immunogen can improve the elicitation of CD4bs bNAbs; ii) long-lasting Env antigen exposure via AAV vector platform can improve the B cell affinity maturation. We will test these hypotheses in small animals. The overall outcome of this study will contribute to our basic understanding of HIV neutralizing antibody responses and the development of a safe and protective HIV vaccine.